Docosahexaenoyl ethanolamides

ABSTRACT

The invention describes novel mono or dihydroxy docosahexaenoic acid (DHA) analogues, their preparation, isolation, identification, purification and uses thereof.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application claims benefit under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/495,705, entitled “DOCOSAHEXAENOYLETHANOLAMIDES”, filed Jun. 10, 2011 (attorney docket numberBWHLP0073US.P1), the contents of which is incorporated herein byreference in it's entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The work leading to this invention was supported in part by NationalInstitutes of Health (NIH) grants R01NS067686, RC2AT005909 andR01DE019938. The U.S. Government therefore may have certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates generally to novel mono and dihydroxy analogues ofdocosahexaenoic acid (DHA) a having a hydroxyl group at C-17 of thecarbon chain or an epoxide at C-16/C-17 and optionally, a secondhydroxyl group at either the C-4, C-7 or C-10 positions of the carbonchain. The present invention also provides novel mono hydroxyl analoguesof docosahexaenoic acid having a hydroxyl group at C-13 with an epoxideat C16/C17, a thiol derivative at C-16 with a hydroxyl at C-17 or adihydroxy compound having hydroxyl groups at C-4 and C-20.

BACKGROUND OF THE INVENTION

Neuroinflammation and local pro-inflammatory mediators are associatedwith neurodegenerative diseases as well as traumatic brain injury (1).In both scenarios, treatment with docosahexaenoic acid (DHA) reducesinflammation and local tissue injury. For example, DHA reduces thedamage from impact acceleration injury and reduces β-amyloid precursor,a marker of axonal injury in vivo relevant in traumatic brain injury(2). Also, DHA reduces ischemic stroke in rats via production ofneuroprotectin D1, which acts on leukocytes and reduces leukocyteinfiltration, leukocyte-mediator tissue damage and regulates NF-κB (3).Neuroprotectin D1 stimulates neuronal stem cell differentiation (4) andhas potent anti-inflammatory and pro-resolving actions in several invivo disease models (5-7). D series resolvins are biosynthesized fromDHA in brain tissue and resolving inflammatory exudates (7,8). ResolvinD1 and resolvin D2 display potent stereoselective actions that areanti-inflammatory, pro-resolving and reduce pain signaling, and act inthe pico- to nanomolar range in vivo, a dose range where DHA itselfdisplays no demonstrable action (9-11). Hence, the metabolome andmetabolic fate of DHA is of interest in the resolution of pain,inflammation and tissue injury.

Another metabolic fate of DHA in brain is conversion to docosahexaenoylethanolamine (DHEA), which is thought to be produced by the same pathwayas N-acyl-arachidonoyl-ethanolamide (AEA, anandamide) (12). DHEA isdirectly related to dietary intake of DHA and is enriched in braintissue at comparable levels to AEA (13). AEA is an endocannabinoid thatregulates neurofunctions and the immune system via CB1 and CB2 receptors(14-17).

Therefore, a need exists for a further understanding of, an explorationor and identification of new useful materials previously not appreciatedas potent biological mediators of interest.

BRIEF SUMMARY OF THE INVENTION

Neuroinflammation and traumatic brain injury involve activation of

inflammatory cells and production of local pro-inflammatory mediatorsthat can amplify tissues damage. Using an LC-UV-MS-MS based lipidomic intandem with functional screening at the single cell level inmicrofluidic chambers, we identified a series of novel bioactiveoxygenated docosahexaenoic ethanolamine-(DHEA) derived products thatregulated leukocyte motility. These included10,17-dihydroxydocosahexaenoyl ethanolamine (10,17-diHDHEA) and15-hydroxy-16(17)-epoxy-docosapentaenoyl ethanolamine (15-HEDPEA), eachof which was an agonist of recombinant CB2 receptors with EC₅₀ 3.9×10⁻¹⁰M and 1.0×10⁻¹⁰ M. In human whole blood, 10,17-diHDHEA and 15-HEDPEA atconcentrations as low as 10 pM each prevented formation ofplatelet-leukocyte aggregates involving either platelet-monocyte orplatelet-PMN. In vivo, 15-HEDPEA was organ protective in mousereperfusion second organ injury. Together these results indicate thatDHEA oxidative metabolism produces potent novel molecules withanti-inflammatory and organ protective properties.

The present invention surprisingly provides novel mono and dihydroxyanalogues of docosahexaenoic acid (DHA) having a hydroxyl group at C-17of the carbon chain or an epoxide at C-16/C-17 and optionally, a secondhydroxyl group at either the C-4, C-7 or C-10 positions of the carbonchain. The present invention also provides novel mono hydroxyl analoguesof docosahexaenoic acid having a hydroxyl group at C-13 with an epoxideat C16/C17, a thiol derivative at C-16 with a hydroxyl at C-17 or adihydroxy compound having hydroxyl groups at C-4 and C-20. Thesematerials are biogenically derived and isolated from media.

In one embodiment, the invention pertains to a new and useful DHAanalogue such as a compound comprising the formula (I):

wherein P₁ is a protecting group or a hydrogen atom;

wherein

is a double bond;

wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)NR^(c)R^(c)—OH, —C(O)H,—C(NH)NR^(c)R^(c), —C(S)H, —C(S)OR^(d), —C(S)NR^(c)R^(c), or —CN;

each R^(a), is independently selected from hydrogen, (C1-C6) alkyl,(C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) arylphenyl (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl,piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 memberedheteroaryl or 6-16 membered heteroarylalkyl;

each R^(c), is independently a protecting group or R^(a), or,alternatively, each R^(c) is taken together with the nitrogen atom towhich it is bonded to form a 5 to 8-membered cycloheteroalkyl orheteroaryl which may optionally include one or more of the same ordifferent additional heteroatoms and which may optionally be substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups;

each R^(b) is independently selected from ═O, —OR^(d), (C1-C3)haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c),halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d),—OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c),—OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d),—[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) or—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each n, independently is an integer from 0 to 3; and

each R^(d), independently is a protecting group or R^(a);

or a pharmaceutically acceptable salt thereof. In one aspect, when Z is—C(O)OR^(d), then R^(d) for Z is not a hydrogen. In certain aspects, P₁is a hydrogen atom. In another aspect, the double bonds at the C-4/5,7/8, 10/11, 13/14 and 19/20 positions are each of Z configuration. Inone aspect, the double bond at C-15/16 is of E configuration.

A particular isomer of interest of the DHA analogue (I) is (Ia)comprising the formula:

wherein P₁,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. In oneembodiment, P₁ is a hydrogen atom.

Another isomer of interest of the DHA analogue (I) is (Ib) comprisingthe formula:

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined. In one embodiment, P₁ is a hydrogen atom.

It should be understood that compounds (I) through (Ib) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect; the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (I)through (Ib). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the invention pertains to a new and useful DHAanalogue such as a compound comprising the formula (I):

wherein P₁,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. P₂,independently of P₁, is a protecting group or a hydrogen atom. In oneaspect, when Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. Inone embodiment, P₁ and P₂ are both hydrogen atoms. In anotherembodiment, the double bonds at the C-7/8, 10/11, 13/14 and 19/20positions are each of Z configuration.

In another aspect, the present invention provides new and useful DHAanalogues such as:

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. In oneaspect, P₁ and P₂ are both hydrogen atoms. In still another aspect, thedouble bonds at C-7/8, 10/11, 13/14 and 19/20 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as:

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined. In one embodiment, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (II) through (IIb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (II)through (IIb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (III):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at the C-4/5, 10/11, 13/14 and 19/20 positions are eachof Z configuration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IIIa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at the C-4/5, 10/11, 13/14 and 19/20 positions are eachof Z configuration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IIIb):

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d), R₂ and n are aspreviously defined. In one aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (III) through (IIIb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (III)through (IIIb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In yet another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (IV):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(c) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In one embodiment, thedouble bonds at C-4/5, 7/8, 13/14 and 19/20 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IVa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at C-4/5, 7/8, 13/14 and 19/20 positions are each of Zconfiguration,

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IVb):

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are aspreviously defined. In one aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (IV) through (IVb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (IV)through (IVb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the invention provides a compound comprising theformula (V):

wherein P₁,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneaspect, P₁ is a hydrogen atom, In another aspect, the double bonds atC-4/5, 7/8, 10/11 and 19/20 positions are each of Z configuration.

In still yet another aspect, the present invention provides a compoundcomprising the formula (Va):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previouslydefined. In one aspect, P₁, is a hydrogen atom. In another aspect, Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom.

It should be understood that compounds (V) and (Va) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (V) and(Va). Purified compounds include that when Z is —C(O)OR^(d), then R^(d)for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VI):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined.

In one aspect, P₁ is a hydrogen atom. In another aspect, Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom. In another embodiment thedouble bonds at the C- 4/5, 7/8, 10/11 and 19/20 positions are each of Zconfiguration.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VIa):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previouslydefined. In one aspect, P₁ is a hydrogen atom.

It should be understood that compounds (VI) and (VIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VI) and(VIa). Purified compounds include that when Z is —C(O)OR^(d), then R^(d)for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VII):

wherein P₁, each Z independently, R^(a), R^(b), R^(c), R^(d) and n areas previously defined.

In one aspect, P₁ is a hydrogen atom. In another aspect, each Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom. In another embodiment,the double bonds at the C-4/5, 7/8 and 19/20 positions are each of Zconfiguration.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VIIa):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previouslydefined. In one aspect, P₁ is a hydrogen atom.

It should be understood that compounds (VII) and (VIIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VII) and(VIIa). Purified compounds include that when Z is —C(O)OR^(d), thenR^(d) for Z is a hydrogen.

In yet another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (VIII):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In one embodiment, thedouble bonds at C-7/8, 13/14 and 16/17 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (VIIIa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (VIII) and (VIIIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VIII)and (VIIIa). Purified compounds include that when Z is —C(O)OR^(d), thenR^(d) for Z is a hydrogen.

In another embodiment, the hydrogen atom of the hydroxyl bearing carbonatom(s), e.g, at —OP₁ and/or OP₂ can be replaced with an alkyl group,such as a methyl group, to help prevent oxidation for any of compounds Ithrough VIIIa.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or more compounds of the invention, with orwithout other active pharmaceutical ingredients, in admixture with apharmaceutically acceptable vehicle. Such a preparation can beadministered according to the methods of the current invention.

In yet another aspect, the present invention is drawn to methods fortreating or preventing inflammation or inflammatory disease in a mammal.The method involves administering a prophylactically or therapeuticallyeffective amount of at least one compound of the invention, or apharmaceutical composition thereof. For example, the compounds of theinvention can be used to treat or prevent inflammation, cancer,neurodegeneration, memory loss, wrinkles, psoriasis, dandruff ordermatitis by administering to an individual in need thereof, aneffective amount of any of the compounds described herein.

Additionally, the compounds of tire invention can be used to neuraldevelopment, fetal development, homeostasis, tissue remodeling, or woundrepair by administering to an individual in need thereof, an effectiveamount of any of the compounds described herein.

Additional features and advantages of the invention will become moreapparent from the following detailed description and claims.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description. As will be apparent, the inventionis capable of modifications in various obvious aspects, all withoutdeparting from the spirit and scope of the present invention.Accordingly, the detailed descriptions are to be regarded asillustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Identification of hydroxydocosahexaenoylethanolamide andfunctional screening of DHEA brain metabolome, a) Online UV, b) tandemmass spectrum of 17-HDHEA. Inset shows fragment assignments for HDHEAmass spectrum, and c) representative average of PMN directionalmigration velocity in 0-10 nM IL-8 gradient (μm/min) before and aftermetabolite mixtures were infused to the chamber. The mixtures wereisolated from mouse brain homogenate incubated with 5 μg DHEA. Errorbars represent migration distance ±S.D. for mean of 26 single PMN (n=3separate donors). *P<0.01 for IL-8 versus brain extracts.

FIG. 2. DHEA metabolome via LC-UV-MS-MS based lipidomics. a-f) OnlineUV, fragment assignments shown in inset, and tandem mass spectrum of4,17-diHDHEA, 7,17-diHDHEA and 10,17-diHDHEA.

FIG. 3. Microfluidic chamber based screening of human PMN chemotaxiswith DHEA derived products, a-c) Representative average PMN directionalmigration displacement against 0-10 nM IL-8 gradient from originalpositions (μm) before and after HPLC isolated DHEA derived products wereindividually infused to the chambers. Insets in a) show morphology ofPMN before (left side) and after (right side) exposure to 10 nM15-HEDPEA (average of 23-30 PMN in each panel).

FIG. 4. GPCR CB1 and CB2 are activated by DHEA-derived products. HEKcells overexpressing CB1 or CB2 in a beta-arrestin system were incubatedwith indicated concentrations of compounds for 1 h in serum free DMEM at37° C. Ligand receptor interactions were determined by increases inchemiluminescence generated upon interaction of the EA labeled betaarrestin with the Pro-Link tagged receptor (see Methods). Dose responseactivation of (a) GPCR CB1 and (b-d) GPCR CB2 with indicated compounds.CB2 receptor-ligand interactions were confirmed with dose response ofCB2 specific inhibitor AM630 coincubated with GPCR C82 over-expressedcell activation stimulated with (e) 15-HEDPEA (10 nM), and (f) AEA (10nM) as positive control. AM630 inhibited GPCR C32 interaction with 10,17S-diHDHEA at higher concentration (data not shown). See text for moredetails.

FIG. 5. In human whole blood, 10,17,S-diHDHEA and 15-HEDPEA each blockPAF-stimulated platelet-leukocyte aggregate formation. Human whole bloodstimulated with PAF (100 nM) was incubated with 10,17-diHDHEA, 15-HEDPEAor DHEA for 30 min, 37° C. The incubation was stopped via ice-cold RBClysis buffer. The majority of RBCs were removed and remaining cells werelabeled. Platelet-leukocyte aggregate formation or P-selectinmobilization was analyzed using FACS (see Methods). Dose responseinhibition of a) platelet-monocyte aggregate formation, b)representative dot plot of platelet-monocyte aggregates, c) platelet-PMNaggregate formation, d) platelet P-selectin mobilization, with indicatedproducts, (diamonds 10,17-diHDHEA; triangles 15-HEDPEA; squares DHEA).Results are mean±SEM of n=5-6 donors. P<0.05 compared to vehicletreatment.

FIG. 6. Proposed DHEA metabolome and bioactive products.

Supplemental FIG. 1. Representative tandem mass and UV spectrum of17-HDHEA prepared from incubations of DHEA with 15-lipoxygenase (seeMethods for details).

Supplemental FIG. 2, DHEA metabolome via LC-UV-MS-MS-based lipidomics.(a-f) tandem mass spectrum and fragmentation assignments, GC-MS massspectrum and dominant ion fragmentation assignments, and selected ionchromatograph of 13-HEDPEA and 15-HEDPEA.

Supplemental FIG. 3. Microfluidic chamber screening of human PMNchemotaxis, (a-c) Representative average PMN directional migrationdisplacement against a 0-10 nM IL-8 gradient. Analyses were carried outas in FIGS. 1 and 3.

Supplemental FIG. 4, 15-HEDPEA is protective in mouse ischemiareperfusion second organ lung injury. Mice were subjected to hind limbischemia for 1 hr using tournequets. 15-HEDPEA was intravenouslyadministered 5 min before tourniquet removal. After 2 hr reperfusion,mouse lungs were harvested and the tissue levels of myeloperoxidase(MPO) were determined using a mouse MPO ELISA. Results are expressed asreduction in lung associated decrease in leukocyte myeloperoxidasevalues (n=3-6).

DETAILED DESCRIPTION

Since AEA undergoes oxidative metabolism to bioactive molecules (16,18),the present invention addressed whether the beneficial actions of DHAtreatment, for example, brain injury(2) can be regulated in part byconversion of DHEA to bioactive products.

The present invention provides the DHEA metabolome with identificationof novel potent bioactive molecules that are organ protective in vivo.These novel bioactive products from DHEA were identified usingLC-MS-MS-based lipidomics in tandem with functional single-cellscreening in newly engineered microfluidic chambers and in vivo systems.These new bioactive products from DHEA may underlie some of thebeneficial effects of DHA administration.

Abbreviations used throughout the specification:

AEA, anandamide, arachidonoylethanolamide;

DHEA, docosahexaenoylethanolamide;

4,17-diHDHEA, 4,17-dihydroxydocosa-5,7Z,10Z,13Z,15,19Z-hexaenoylethanolamide;

7,17-diHDHEA,7,17-dihydroxydocosa-4Z,8E,10Z,13Z,15,19Z-hexaenoylethanolamide;

10, 17-diHDHEA,10,17-dihydroxydocosa-4Z,7Z,11,13Z,15,19Z-hexaenoylethanolamide;

17-HDHEA, 17-hydroxydocosa-4Z,7Z, 10Z,13Z,15,19Z-hexaenoylethanolamide;

13-HEDPEA, 13-hydroxy-16(17)-epoxydocosa-4Z,7Z,10Z,14,19Z-pentaenoylethanolamide;

15-HEDPEA,15-hydroxy-16(17)-epoxydocosa-4Z,7Z,10Z,13Z,19Z-pentaenoylethanolamide;

LOX, lipoxygenase, abstracts hydrogen and inserts molecular oxygen in astereoselective reaction with 1,4-cis-pentadiene units present inpolyunsaturated fatty acids.

In the specification and in the claims, the terms “including” and“comprising” are open-ended terms and should be interpreted to mean“including, but not limited to . . . .” These terms encompass the morerestrictive terms “consisting essentially of” and “consisting of.”

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”,“characterized by” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. All publications and patentsspecifically mentioned herein are incorporated by reference in theirentirety for all purposes including describing and disclosing thechemicals, instruments, statistical analyses and methodologies which arereported in the publications which might be used In connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

“Compounds of the invention” refers to the mon-hydroxy, di-hydroxy,and/or epoxide DHA analogues and compounds encompassed by genericformulae disclosed herein and includes any specific compounds withinthose formulae whose structure is disclosed herein. The compounds of theinvention may be identified either by their chemical structure and/orchemical name. When the chemical structure and chemical name conflict,the chemical structure is determinative of the identity of the compound.The compounds of the invention may contain one or more chiral centersand/or double bonds and therefore, may exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, the chemical structures depicted hereinencompass all possible enantiomers and stereoisomers of the illustratedcompounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomer mixtures. Enantiomeric andstereoisomeric mixtures can be resolved into their component enantiomersor stereoisomers using separation techniques or chiral synthesistechniques well known to the skilled artisan. The compounds of theinvention also include isotopically labeled compounds where one or moreatoms have an atomic mass different from the atomic mass conventionallyfound in nature.

The compounds depicted throughout the specification containethylenically unsaturated sites. Where carbon carbon double bonds exist,the configurational chemistry can be either cis (Z) or trans (E) and thedepictions throughout the specification are not meant to be limiting.The depictions are, in general, presented based upon the configurationalchemistry of related DHA compounds, and although not to be limited bytheory, are believed to possess similar configuration chemistry. The useof

reflects this throughout the specification and claims so that both cisand trans isomers are contemplated. In certain embodiments theconfiguration of the ethylenic bond is known and is particularlydescribed.

In one aspect of the invention, the compound(s) of the invention aresubstantially purified and/or isolated by techniques known in the art.The purity of the purified compounds is generally at least about 90%,preferably at least about 95%, and most preferably at least about 99% byweight.

Thus, the term “purified” as used herein does not require absolutepurity; rather, it is intended as a relative term. For example, apurified DHA analogue can be one in which the subject DHA analogue is ata higher concentration than the analogue would be in its naturalenvironment within an organism. For example, a DHA analogue, of theinvention can be considered purified if the analogue content in thepreparation represents at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,98%, or 99% of the total analogue content of the preparation.

“Biological activity” and its contextual equivalents “activity” and“bioactivity” means that a compound elicits a statistically valid effectin any one biological test assays. Preferably, the threshold fordefining an “active” compound will be reproducible and statisticallyvalid effects of at least 25% deviation from untreated control atconcentrations at or lower than 1 μM.

“Biological test assay” means a specific experimental procedure.Non-limiting examples of biological, test assays include: 1) ligandbinding, either direct or indirect, to a purified target, subcellularfraction, intact cell, or cell or tissue extract; 2) metabolicprotection with enhanced half-life when exposed to a purified target,subcellular fraction, intact cell, cell or tissue extract, oradministered to intact organism by any route; 3) prevention, reversal,or amelioration of cell- and tissue-based functional responsesrecognized by skilled artisans to represent surrogates foranti-inflammatory action (e.g., altered cytokine production andrelease); and 4) prevention, reversal, or amelioration of symptomsand/or disease processes in animal models of inflammation andinflammatory disease.

“Detectable label” means any chemical or biological modality which canbe used to track, trace, localize, quantify, immobilize, purify, oridentify compounds through appropriate means of detection known in theart. Non-limiting examples of detectable labels include fluorescence,phosphorescence, luminescence, radioactive or biospecific affinitycapture labels.

“Electronegative group” is a chemical group that tends to acquire ratherthan lose electrons in its chemical interactions. Examples ofelectronegative groups include, but are not limited to, —NO₂, ammoniumsalts, sulfonyl groups, carbonyl groups, halogens, esters, carboxylicacids, nitrites, etc.

“In Situ” refers to and includes the terms “in vivo,” “ex vivo” and “invitro” as these terms are commonly recognized and understood by theskilled artisan. Moreover, the phrase “in situ” is employed herein, inits broadest connotative and denotative context to identify an entity,cell, or tissue as found or in place, without regard to its source ororigin, its condition or status or its duration or longevity at thatlocation or position.

“Pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopoeia orother generally recognized pharmacopoeia for use in animals, and moreparticularly in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. Such saltsinclude: (1) salts formed when an basic proton is present in the parentcompound such as acid addition salts, formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or those formed with organic acids suchas acetic acid, propionic acid, hexanoic acid, cyclopentanepropionicacid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonicacid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid and the like; or (2)salts formed when an acidic proton is present in the parent compound andeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase such as ethanolamine, diethanolamine, triethanolamine,N-methylglucamine, triethylamine, propylamino, diazabicycloundecane andthe like.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a compound(s) of thepresent invention within or to the subject such that it can perform itsintended function. Typically, such compounds are carried or transportedfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; tale; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations.

“Prodrug” refers to a derivative of a drug molecule that requires a

transformation within the body to release the active drug. Prodrugs arefrequently (though not necessarily) pharmacologically inactive untilconverted to the parent drug. A hydroxyl containing drug may beconverted to, for example, to a sulfonate, ester or carbonate prodrug,which may be hydrolyzed in vivo to provide the hydroxyl compound. Anamino containing drug may be converted, for example, to a carbamate,amide, imine, phosphonyl, phosphoryl or sulfenyl prodrug, which may behydrolyzed in vivo to provide the amino compound. A carboxylic acid drugmay be converted to an ester (including silyl esters and thioesters),amide or hydrazide prodrug, which be hydrolyzed in vivo to provide thecarboxylic acid compound. Prodrugs for drugs which contain differentfunctional groups other than those listed above are well known to theskilled artisan.

“Promoiety” refers to a form of protecting group that when used to maska functional group within a drug molecule converts the drug into aprodrug. Typically, the promoiety will be attached to the drug viabond(s) that are cleaved by enzymatic or non-enzymatic means in vivo.

“Protecting group” refers to a grouping of atoms that when attached to areactive functional group in a molecule masks, reduces or preventsreactivity of the functional group. Examples of protecting groups can befound in Green et al., “Protective Groups in Organic Chemistry”, (Wiley,2.sup.nd ed. 1991) and Harrison et al,, “Compendium of Synthetic OrganicMethods,” Vols. 1-8 (John Wiley and Sons, 1971-1996), Representativeamino protecting groups include, but are not limited to, formyl, acetyl,trifluoroacetyl, benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl(“Boc”), trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl(“SES”), trityl and substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxy protecting groups include,but are not limited to, those where the hydroxy group is either acylated(e.g., methyl and ethyl esters, acetate or propionate groups or glycolesters) or alkylated such as benzyl, and trityl ethers as well as alkylethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS orTIPPS groups) and allyl ethers.

“Subject” means living organisms susceptible to conditions or diseasescaused or contributed to by inflammation, inflammatory responses,vasoconstriction and myeloid suppression. Examples of subjects includehumans, dogs, eats, cows, goats and mice. The term subject is furtherintended to include transgenic species such as, for example, transgenicmice.

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom irons a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cvcloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C1-C6) alkyl,

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like. Inpreferred embodiments, the alkanyl groups ate (C1-C6) alkanyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon, atom of a parent alkene. The group may be ineither the cis or trans conformation about the double bond(s). Typicalalkenyl groups include, but are not limited to, ethenyl; propenyls suchas prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc.;and the like. In preferred embodiments, the alkenyl group is (C2-C6)alkenyl.

“Alkynyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc,; and the like. In preferredembodiments, the alkynyl group is (C2-C6) alkynyl.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or

unsaturated, branched, straight-chain or cyclic divalent hydrocarbongroup having the stated number of carbon atoms (i.e., C1-C6 means fromone to six carbon atoms) derived by the removal of one hydrogen atomfrom each of two different carbon atoms of a parent alkane, alkene oralkyne, or by the removal of two hydrogen atoms from a single carbonatom of a parent alkane, alkene or alkyne. The two monovalent radicalcenters or each valency of the divalent radical center can form bondswith the same or different atoms. Typical alkyldiyl groups include, butare not limited to, methandiyl; ethyldiyls such as ethan-1,1-diyl,ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl; propyldiyls such aspropan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl,cyclopropan-1,1-diyl, cyclopropan-1,2-diyl, prop-1-en-1,1-diyl,prop-1-en-1,2-diyl, prop-2-en-1,2-diyl, prop-1-en-1,3-diyl,cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl,butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1 -diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien1,4-diyl,cyclobut-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl.cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl. etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In preferredembodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also preferredare saturated acyclic alkanyldiyl groups in which the radical, centersare at the terminal carbons, e.g., methandiyl (methano); ethan-1,2-diyl(ethane); propan-1,3-diyl (propano); butan-1,4-diyl (butane); and thelike (also referred to as alkylenos, defined infra).

“Alkdiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (i.e., C1-C6means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typical alkdiylgroups include, but are not limited to methandiyl; ethyldiyls such asethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl- , prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl,butan-1,4-diyl, butan-2,2-diyl, 2-methyl-propan-1,1-diyl,2-methyl-propan-1,2-diyl, cyclobutan-1,1-diyl; cyclobutan-1,2-diyl,cyclobutan-1,3-diyl, but-1-en-1,1-diyl, but-1-en-1,2-diyl,but-1-en-1,3-diyl, but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1 -diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.: andthe like. Where specific levels of saturation are intended, thenomenclature alkandiyl, alkendiyl and/or alkyndiyl is used. In apreferred embodiment, the alkdiyl group is (C1-C6) alkdiyl. Alsopreferred are saturated acyclic alkanyldiyl groups in which the radicalcenters are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenes, defined infra)

“Alkyleno” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkyleno is indicatedin square brackets. Typical alkyleno groups include, but are not limitedto, methano; ethylenes such as ethano, etheno, ethyno; propylenes suchas propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenossuch as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkano, alkeno and/or alkynois used. In preferred embodiments, the alkyleno group is (C1-C6) or(C1-C3) alkyleno. Also preferred are straight-chain saturated alkanogroups, e.g., methano, ethano, propano, butano, and the like.

“Heteroalkyl,” “Heteroalkanyl,” “Heteroalkenyl,” “Heteroalkynyl,”“Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteratoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR′—, —PH—, —S(O)₂—, —S(O)₂—, —S(O)NR′—, —S(O)₂NR′—, and the like, including combinations thereof, whereeach R′ is independently hydrogen or (C1-C6) alkyl.

“Cycloalkyl” and “Heterocycloalkyl” by themselves or as part of anothersubstituent refer to cyclic versions of “alkyl” and “heteroalkyl”groups, respectively. For heteroalkyl groups, a heteroatom can occupythe position that is attached to the remainder of the molecule. Typicalcycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. Typical heterocycloalkyl groups include, butare not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), and the like.

“Acyclic Heteroatomic Bridge” refers to a divalent bridge in which thebackbone atoms are exclusively heteroatoms and/or heteroatomic groups.Typical acyclic heteroatomic bridges include, but are not limited to,—O—, —S—, —S—O—, —NR′—, —PH—, —S(O)—, —S(O)₂—, —S(O) NR′—, —S(O)₂NR′—,and the like, including combinations thereof, where each R′ isindependently hydrogen or (C1-C6) alkyl.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated π electron system.Specifically included within the definition of “parent aromatic ringsystem” are fused ring systems in which one or more of the rings arearomatic and one or more of the rings are saturated or unsaturated, suchas, for example, fluorene, indane, indene, phenalene,tetrahydronaphthalene, etc. Typical parent aromatic ring systemsinclude, but are not limited to, aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azolene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, oetalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and thelike, as well as the various hydro isomers thereof.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In preferred embodiments, the aryl groupis (C5-C15) aryl, with (C5-C10) being even more preferred. Particularlypreferred aryls are cyclopentadienyl, phenyl and naphthyl.

“Arylaryl” by itself or as part of another substituent refers to amonovalent hydrocarbon group derived by the removal of one hydrogen atomfrom a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C5-C15) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parentaromatic ring system of an arylaryl group is independently a (C5-C15)aromatic, more preferably a (C5-C10) aromatic. Also preferred arearylaryl groups in which all of the parent aromatic ring systems areidentical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

“Biaryl” by itself or as part of another substituent refers to anarylaryl group having two identical parent aromatic systems joineddirectly together by a single bond. Typical biaryl groups include, butare not limited to, biphenyl, binaphthyl, bianthracyl, and the like.Preferably, the aromatic ring systems are (C5-C15) aromatic rings, morepreferably (C5-C10) aromatic rings. A particularly preferred biarylgroup is biphenyl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). Inparticularly preferred embodiments the arylalkyl group is (C6-C13),e.g., the alkanyl, alkenyl or alkynyl moiety of the aryiaikyl group is(C1-C3) and the aryl moiety is (C5-C10).

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, benzodioxan, benzofuran, chromane, chromene,indole, indoline, xanthene, etc. Also included in the definition of“parent heteroaromatic ring system” are those recognized rings thatinclude common substituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Typical parent heteroaromatic ring systemsinclude, but are not limited to, acridine, benzimidazole, benzisoxazole,benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole,benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline,carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole,indazole, indole, indoline, indolizine, isobenzofuran, isochromene,isoindole, isoindoline, isoquinoline, isothiazole, isoxazole,naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine,phenanthroline, phenazine, phthalazine, pteridine, purine, pyran,pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizinc, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike.

“Heteroaryl” by itself or as part of another substituent refers to a

monovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In preferredembodiments, the heteroaryl group is a 5-14 membered heteroaryl, with5-10 membered heteroaryl being particularly preferred.

“Heteroaryl-Heteroaryl” by itself or as part of another substituentrefers to a monovalent heteroaromatic group derived by the removal ofone hydrogen atom from a single atom of a ring system in which two ormore identical or non-identical parent heteroaromatic ring systems arejoined directly together by a single bond, where the number of suchdirect ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridyipurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-15 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical.

“Biheteroaryl” by itself or as part of another substituent refers to aheteroaryl-heteroaryl group having two identical parent heteroaromaticring systems joined directly together by a single bond. Typicalbiheteroaryl groups include, but are not limited to, bipyridyl,bipurinyl, biquinolinyl, and the like. Preferably, the heteroaromaticring systems are 5-15 membered heteroaromatic rings, more preferably5-10 membered heteroaromatic rings.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In particularlypreferred embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen, atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR″, “alkylamine” refers to a group of the formula—NHR″ and “dialkylamine” refers to a group of the formula —NR″R″, whereeach R″ is independently an alkyl. As another example, “haloalkoxy” or“haloalkyloxy” refers to a group of the formula —OR″′, where R″′ is ahaloalkyl.

The present invention is also drawn to methods for treating arterialinflammation, arthritis, psoriasis, urticara, vasculitis, asthma, ocularinflammation, pulmonary inflammation, pulmonary fibrosis, seborrheicdermatitis, pustular dermatosis, or cardiovascular diseases in a subjectby administration of one or more of the DHA analogs described herein.Disease states or conditions that are associated with inflammation suchas the recruitment of neutrophils, leukocytes and/or cytokines areincluded within the general scope of inflammation and include, forexample, Addiction, AIDS, Alcohol-related disorders, Allergy,Alzheimer's disease, Anesthesiology, Anti-infectives, Anti-inflammatoryagents. Arthritis, Asthma, Atherosclerosis, Bone diseases, Breastcancer, Cancer, Cardiovascular diseases, Child health, Colon, cancer,Congenital defects, Decision analysis, Degenerative neurologicdisorders, Dementia, Dermatology, Diabetes mellitus, Diagnostics, Drugdelivery, Drug discovery/screen, Endocrine disorders, ENT, Epidemiology,Eye diseases, Fetal and maternal medicine, Gastrointestinal disorders,Gene therapy, Genetic diagnostics, Genetics, Genitourinary disorders,Geriatric medicine, Growth and Development, Hearing, Hematologicdisorders, Hepatobiliary disorders, Hypertension, imaging, Immunology,Infectious diseases, Leukemia/lymphoma, Lung cancer, Metabolicdisorders, Neonatology, Neurological disorders, Neuromusculardisorder's, Nuclear medicine, Obesity/eating disorders, Orthopedic,Other, Parasitic diseases, Perinatal disorders, Pregnancy, Preventativemedicine, Prostate cancer, Psychiatric disorders, Pulmonary disorders,Radiology, Renal disorders, Reproduction, Rheumatic diseases, Stroke,Surgical, Transplantation, Vaccines, Vascular medicine, Wound healing,oral infections, periodontal disease, brain injury, trauma and neuronalinflammation, and Women's health.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the DHA analogs of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result, e.g., a diminishment or prevention of effectsassociated with various disease states or conditions. A therapeuticallyeffective amount of the DHA analog may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the therapeutic compound to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the therapeutic agent, are outweighed bythe therapeutically beneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the DHA analog and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a DHA analog of the invention is0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time disordersto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient, i.e., at least one DHAanalog, in combination with a pharmaceutically acceptable carrier.

In certain embodiments, the compounds of the present invention maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts, esters,amides, and prodrugs” as used herein refers to those carboxylate salts,amino acid addition salts, esters, amides, and prodrugs of the compoundsof the present invention which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of patientswithout undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use of the compounds of the invention. The term “salts”refers to the relatively non toxic, inorganic and organic acid additionsalts of compounds of the present invention. These salts can be preparedin situ during the final isolation and purification of the compounds orby separately reacting the purified compound in its free base form witha suitable organic or inorganic acid and isolating the salt thus formed.These may include cations based on the alkali and alkaline earth metals,such as sodium, lithium, potassium, calcium, magnesium and the like, aswell as non toxic ammonium, quaternary ammonium, and amine cationsincluding, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like, (See, for example, Berge S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977;66:1 19 which isincorporated herein by reference).

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterifies products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its tree acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. The term isfurther intended to include lower hydrocarbon groups capable of beingsolvated under physiological conditions, e.g., alkyl esters, methyl,ethyl and propyl esters.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfite, sodium metabisulfite, sodium sulfite and tie like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable forintravenous, oral, nasal, topical, transdermal, buccal, sublingual,rectal, vaginal and/or parenteral administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect. Generally, out of one hundred per cent,this amount will range from about 1 per cent to about ninety-ninepercent of active ingredient, preferably from about 5 per cent to about70 per cent, most, preferably from about 10 per cent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessor/ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: filler's or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; humectants, such as glycerol,disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;solution retarding agents, such as paraffin; absorption accelerators,such as quaternary ammonium compounds; wetting agents, such as, forexample, cetyl alcohol and glycerol monostearate; absorbents, such askaolin and bentonite clay; lubricants, such a talc, calcium stearate,magnesium, stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin, or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which cars be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, com, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention torrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, sad which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellents, such,as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention. Suchsolutions are useful for the treatment of conjunctivitis.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered dragform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Intravenous injection administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systematically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient'system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of ordinary skill in the art. Actual dosage levels of theactive ingredients in the pharmaceutical compositions of this inventionmay be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated analgesic effects, will range from about0.0001 to about 100 mg per kilogram of body weight per day, morepreferably from about 0.01 to about 50 mg per kg per day, and still morepreferably from about 0.1 to about 40 mg per kg per day. For example,between about 0.01 microgram and 20 micrograms, between about 20micrograms and 100 micrograms and between about 10 micrograms and 200micrograms of the compounds of the invention are administered per 20grams of subject weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The invention features an article of manufacture that contains packagingmaterial and DHA analog formulation contained within the packagingmaterial. This formulation contains an at least one DHA analog and thepackaging material contains a label or package insert indicating thatthe formulation can be administered to the subject to treat one or moreconditions as described herein, in an amount, at a frequency, and for aduration effective to treat or prevent such condition(s). Suchconditions are mentioned throughout the specification and areincorporated herein by reference. Suitable DHA analogs are describedherein.

The present invention surprisingly provides novel mono and dihydroxyanalogues of docosahexaenoic acid (DHA) having a hydroxyl group at C-17of the carbon chain or an epoxide at C-16/C-37 and optionally, a secondhydroxyl group at either the C-4, C-7 or C-10 positions of the carbonchain. The present invention also provides novel mono hydroxyl analoguesof docosahexaenoic acid having a hydroxyl group at C-13 with an epoxideat C16/C17, a thiol derivative at C-16 with a hydroxyl at C-17 or adihydroxy compound having hydroxyl groups at C-4 and C-20. Thesematerials are biogenically derived and isolated from media.

In one embodiment, the invention pertains to a new and useful DHAanalogue such as a compound comprising the formula (I):

wherein P₁ is a protecting group or a hydrogen atom;

wherein

is a double bond;

wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)NR^(c)R^(c)—OH, —C(O)H,—C(NH)NR^(c)R^(c), —C(S)H, —C(S)OR^(d), —C(S)NR^(c)R^(c), or —CN;

each R^(a), is independently selected from hydrogen, (C1-C6) alkyl,(C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl,phenyl, (C6-C16) arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8membered cycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl,piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 memberedheteroaryl or 6-16 membered heteroarylalkyl;

each R^(c), is independently a protecting group or R^(a), or,alternatively, each R^(c) is taken together with the nitrogen atom towhich it is bonded to form a 5 to 8-membered cycloheteroalkyl orheteroaryl which may optionally include one or more of the same ordifferent additional heteroatoms and which may optionally be substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups;

each R^(b) is independently selected from ═O, —OR^(d), (C1-C3)haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d), —NR^(c)R^(c),halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a), —C(NOH)NR^(c)R^(c), —OC(O)R^(d),—OC(O)OR^(d), —OC(O)NR^(c)R^(c), —OC(NH)NR^(c)R^(c),—OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d), —[NR^(a)C(O)]_(n)R^(d),—[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d), —[NHC(O)]_(n)NR^(c)R^(c),—[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) or—[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c);

each n, independently is an integer from 0 to 3; and

each R^(d), independently is a protecting group or R^(a);

or a pharmaceutically acceptable salt thereof. In one aspect, when Z is—C(O)OR^(d), then R^(d) for Z is not a hydrogen. In certain aspects, P₁is a hydrogen atom. In another aspect, the double bonds at the C-4/5,7/8, 10/11, 13/14 and 19/20 positions are each of Z configuration. Inone aspect, the double bond at C-15/16 is of E configuration.

A particular isomer of interest of the DHA analogue (I) is (Ia)comprising the formula:

wherein P₁,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. Incertain aspects, P₁ is a hydrogen atom.

Another isomer of interest of the DHA analogue (I) is (Ib) comprisingthe formula:

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined. In certain aspects, P₁ is a hydrogen atom.

It should be understood that compounds (I) through (Ib) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (I)through (Ib). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the invention pertains to a new and useful DHAanalogue such as a compound comprising the formula (II):

wherein P₁,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. P₂,independently of P₁, is a protecting group or a hydrogen atom. In oneaspect, when Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. Inone embodiment, P₁ and P₂ are both hydrogen atoms. In anotherembodiment, the double bonds at the C-7/8, 10/11, 13/14 and 19/20positions are each of 2 configuration.

In another aspect, the present invention provides new and useful DHAanalogues such as:

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined. In oneaspect, P₁ and P₂ are both hydrogen atoms, In still another aspect, thedouble bonds at C-7/8, 10/11, 13/14 and 19/20 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as:

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined. In one embodiment. P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (II) through (IIb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (II)through (IIb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (III):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at the C-4/5, 10/11, 13/14 and 19/20 positions are eachof Z configuration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IIIa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at the C-4/5, 10/11, 13/14 and 19/20 positions are eachof Z configuration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IIIb):

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d), R₂ and n are aspreviously defined. In one aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (III) through (IIIb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (III)through (IIIb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In yet another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (IV):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In one embodiment, thedouble bonds at C-4/5, 7/8, 13/14 and 19/20 positions are each ox Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IVa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms. In another embodiment,the double bonds at C-4/5, 7/8, 13/14 and 19/20 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (IVb):

wherein P₁, P₂, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are aspreviously defined. In one aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (IV) through (IVb) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (IV)through (IVb). Purified compounds include that when Z is —C(O)OR^(d),then R^(d) for Z is a hydrogen.

In another aspect, the invention provides a compound comprising theformula (V):

wherein P₁,

, Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined,provided when Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. Inone aspect, P₁ is a hydrogen atom. In another aspect, the double bondsat C-4/5, 7/8, 10/11 and 19/20 positions are each of Z configuration.

In still yet another aspect, the present invention provides a compoundcomprising the formula (Va):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previouslydefined. In one aspect, P₁, is a hydrogen atom. In another aspect, Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom.

It should be understood that compounds (V) and (Va) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (V) and(Va). Purified compounds include that when Z is —C(O)OR^(d), then R^(d)for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VI):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d) and n are as previouslydefined.

In one aspect, P₁ is a hydrogen atom. In another aspect, Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom. In another embodiment,the double bonds at the C-4/5, 7/8, 10/11 and 19/20 positions are eachof Z configuration.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VIa):

wherein P₁, Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previouslydefined. In one aspect, P₁ is a hydrogen atom.

It should be understood that compounds (VI) and(VIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VI) and(VIa). Purified compounds include that when Z is —C(O)OR^(d), then R^(d)for Z is a hydrogen.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VII):

wherein P₁,each Z independently, R^(a), R^(b), R^(c), R^(d) and n are aspreviously defined.

In one aspect, P₁ is a hydrogen atom. In another aspect, each Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom. In another embodiment,the double bonds at the C-4/5, 7/8 and 19/20 positions are each of Zconfiguration.

In another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising the formula (VIIa):

wherein P₁,each Z independently, R^(a), R^(b), R^(c), R^(d), R₁ and nare as previously defined. In one aspect, P₁ is a hydrogen atom.

It should be understood that compounds (VII) and(VIIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VII) and(VIIa). Purified compounds include that when Z is —C(O)OR^(d) , thenR^(d) for Z is a hydrogen.

In yet another aspect, the present invention provides new and useful DHAanalogues such as a compound comprising formula (VIII):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d) and n are as previously defined, providedwhen Z is —C(O)OR^(d), then R^(d) for Z is not a hydrogen. In oneembodiment, P₁ and P₂ are both hydrogen atoms. In one embodiment, thedouble bonds at C-7/8, 13/14 and 16/17 positions are each of Zconfiguration.

In still another aspect, the present invention provides new and usefulDHA analogues such as a compound comprising the formula (VIIIa):

wherein P₁, P₂,

Z, R^(a), R^(b), R^(c), R^(d), R₁ and n are as previously defined. Inone aspect, P₁ and P₂ are both hydrogen atoms.

It should be understood that compounds (VIII) and (VIIIa) include allpharmaceutically acceptable salts, esters thereof, the purified/isolatedforms, as well as compounds wherein the hydroxyl is converted into aprotecting group as described herein.

In another aspect, the present invention provides new and useful DHAanalogues such as a purified compounds comprising the formulae (VIII)and (VIIIa). Purified compounds include that when Z is —C(O)OR^(d), thenR^(d) for Z is a hydrogen.

In another embodiment, the hydrogen atom of the hydroxyl bearing carbonatom(s), e.g. at —OP₁ and/or OP₂ cart be replaced with an alkyl group,such as a methyl group to help prevent oxidation for any of compounds Ithrough VIIIa.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or more compounds of the invention, with orwithout other active pharmaceutical ingredients, in admixture with apharmaceutically acceptable vehicle. Such a preparation can beadministered according to the methods of the current invention.

It should be understood that the intermediates described herein are alsoincluded as part of the invention and can be considered active agents aswell. For example, ketone containing intermediates are within the scopeof the active agents as well as alkyne intermediates as describedherein.

Results and Discussion

While AEA functions as a cannabinoid receptor agonist and its metabolismis well appreciated (12,14-16,27), the roles of DHEA and its metabolomeare of interest since DHA treatment reduces traumatic brain injury (2)and is the precursor to potent proresolving mediators, including (heresolvins and protecting (1,7,8). In the present study, HDHEA wasidentified in mouse brain, which provided the basis for furtherinvestigation of 17-HDHEA and 17-HpDHEA metabolic fates and potentialbiological impact of DHEA metabolism. Given the lack of functionalgroups for efficient ionization via electrospray ionization (ESI),direct analysis/detection of DHEA or its oxygenated metabolites withLC-MS-MS was impeded with low sensitivity. To this end, their acetateadducts, [M+CH₃COOH—H], were targeted for analysis, which proved to be auseful alternative strategy employed in the present investigation. Interms of both detection limits and tandem mass fragmentation patterns,these oxygenated DHEA acetate adducts were comparable to those ox thecorresponding free acid-derived products.

Since AEA is a reported substrate for murine leukocyte type 12/15-LOX,reticulocyte type 15-LOX and soybean 15-LOX to generate15-hydroperoxyarachidonoyl ethanolamine(18), it was rationalized57-HDHEA as the reduced hydroxyl group containing product of15-lipoxygenase-like-enzyme with DHEA. This hypothesis proved consistentwith LC-MS-MS mass analysis of the reduced product obtained fromincubation of DHEA with soy bean 15-LOX, which essentially showed thesame LC retention time, tandem mass fragmentation patterns as well asonline UV spectrum with endogenous 17-HDHEA (FIG. 1).

To determine 17-HpDHEA/17-HDHEA metabolic fates, LC-MS-MS-basedlipidomic investigation led to identification of a series of noveloxygenated products listed in Table 1. Incubation of either 17-HpDHEA orDHEA with human PMN or mouse brain also gave a novel series ofdioxygenated products, such as 4,17-diHDHEA, 7,17-diHDHEA,10,17-diHDHEA, as well as 15-HEDPEA (Table 1). From DHA, some of theseproducts are biosynthesized in inflammatory exudates, namely resolvin D5(7,17-diHDHA) and resolvin D6 (4,17-diHDHA)(8) as well as the doubledioxygenation product 10,17-diHDHA, an isomer of neuroprotectin D1 (6).Hence their ethanolamide counterparts were identified in the presentstudy. In addition, incubation of 17-HpDHEA with hemoglobin generatedtwo major hepoxilin-like structures (29), 13-HEDPEA and 15-HEDPEA. Giventhat hepoxilin diastereomer mixtures are generated from hemoglobin orhemin (29), it was of interest whether this was the ease for17-HpDHEA-derived compounds. To this end, NMR chemical shift of H-18 ofisolated 13-HEDPEA displayed two distinguishable peaks at 4.23 ppm and4.45 ppm, and chemical shift of H-13 of isolated 13-HEDPEA showed broadpeaks around 3.9 ppm (Supplemental Table 2a and 2b), which stronglysuggested the presence of diastereomers. In order to determine thebiosynthetic mechanism of formation of 13-HEDPEA and 15-HEDPEA fromhemoglobin and 17-HpDHEA, incubations were also carried out in ¹⁸Owater, Tandem mass analysis of these incubation products indicated that¹⁸O was not incorporated within these products (data not shown), whichsuggested the oxygen source of hydroxyl group could be attributed toatmospheric O₂.

Table 1 Structures, LC-MS and GC-MS fragmentations, and UV λmax fornovel DHEA metabolites identified using mediator-based lipidomics LCLC-MS reten- major/ PMN + Mouse tion disgnostic UV DHEA brain Trivialtime fragmentions λ max or Hgb + + Structure† Name (min) (m/z) (nm)HpDHEA HpDHEA DHEA

17- HDHEA 26.6 446(M + AcOH—H), 386(M—H), 368, 317, 288, 281, 201. 237yes yes yes

4, 17- diHDHEA 20.6 462(M + AcOH—H), 402(M—H), 384, 366, 333, 315, 304,286, 257, 239, 114 238 yes not detected yes

7, 17- diHDHEA 18.2 462(M + AcOH—H), 402(M—H), 384, 366, 304, 286, 184,156. 246 yes not detected trace

10, 17- diHDHEA 17.7 462(M + AcOH—H), 402(M—H), 384, 366, 304, 196. 270yes not detected trace

15- HEDPEA 21.3 462(M + AcOH—H), 402(M—H), 384, 366, 315, 304 286, 262.conju- gated dou- ble bond sys- tem not pre- sent yes not detected mi-nor, no clear mass spec- trum †Stereochemistries shown are tentativeassignments.

Combining results from lipodomic analyses and the mechanisms proposedfor phytooxylipin and hepoxilin biosynthesis (30), the pathways fornovel oxygenated DHEA products are proposed in FIG. 6. In this scheme,DHEA is first converted to 17-HpDHEA mediated by 15-LOX. Then 17-HpDHEAis partially reduced to the oxide radical (FIG. 6) by hemoglobin, whichreacts with vicinal double bond at 16-position to yield 16(17)-epoxideradical. Non- or low-stereospecific addition of oxygen to intermediateleads to formation of two types of peroxide radical diastcreomers.Further reduction can generate 13-HEDPEA and 15-HEDPEA. Alternatively.17-HpDHEA can undergo reduction to yield 17-HDHEA. Of interest,hemoglobin interactions with 17-HpDHEA yield approximately equal amountof 13-HEDPEA and 15-HEDPEA; for comparison, incubation of PMN with DHEAor HpDHEA generated predominantly 15-HEDPEA (Table 1 and SupplementalTable 1), suggesting the presence of a distinct 15-HEDPEA synthase inhuman PMN.

In view of the requirement for methodology development for functionalscreening to keep up with the rapid expansion of modern metabolomics,microfluidic chambers were coupled in tandem for screening chemotacticactivity of human PMN with the novel DHEA derived products. Given the ˜1μl volume of the assay chamber, only small amounts of materials wererequired for these analyses. Results from the screening reported in FIG.3 indicated that 15-HEDPEA (10 nM) effectively stopped PMN chemotaxisstimulated with IL-8 gradient. Microfluidic chamber-based screening ofhuman PMN chemotaxis offers several advantages that include: a) thesmall amounts needed in the ˜1 μl³ chamber, b) the system permitscapture of human leukocytes in less than five minutes compared toseveral hours (2-3) of isolation using density gradient, and c) videodocumentation of single PMN responses (9). Hence, the present resultsfurther demonstrate microfluidic chamber-based functional screening asan effective novel approach to decode rare and transient functionalmetabolites.

AEA exerts a wide range of functions via binding to CB receptors(14-17). However, its DHA metabolite DHEA displays only moderateaffinity to CB1 receptor (Ki 324 nM vs. 40 nM for AEA) (31). Toinvestigate the biological implications of DHEA metabolic oxidation interms of activating CB receptors, two of the major PMN products wereassessed, 10, 1.7-diHDHEA and 15-HEDPEA, using CB2-beta arrestin ligandsystems. The EC₅₀ for the novel DHEA-derived products, 10,17-diHDHEA and15-HEDPEA, were 3.9×10⁻¹⁰ M and 1.0×10⁻¹⁰ M respectively, similar tothat of AEA (FIG. 4). For comparison, EC₅₀ for DHEA was 9.8×10⁻⁹ M,approximately 2 orders of magnitude higher, 10,17-diHDHEA and 15-HEDPEAalso activated CB1 as shown in FIG. 4a . Ligand-CB2 interactions wereconfirmed using the specific CB2 antagonist AM630 (FIG. 4e,f ). Anothermolecular target of AEA are the vanilloid receptors (TRPVl) in additionto the cannabinoid receptors, which required micromolar range foractivity (32). The results indicated that metabolic oxygenation of DHEAproduces novel CB agonists with enhanced potencies that are in thenanomolar range.

Since the production of certain NAE is enhanced during stroke (33), itwas of interest to investigate biological functions of DHEA and itsmetabolites in platelet-leukocyte aggregate formation in human wholeblood. Platelet-leukocyte aggregate formation is a component of manyvascular diseases, stroke, diabetes, and hypertension (28).Specifically, increased platelet-leukocyte aggregates were suggested asan early marker for acute myocardial infarction and is increasinglyregarded as a cardiovascular risk factor (34). Also patients with,elevated circulating platelet-monocyte aggregates may reflect apro-atherogenic phenotype (35). The presence of platelet-leukocyteaggregates stimulate production of pro-inflammatory cytokines, such asIL-1β, IL-8, MCP-1, MIP-1b, PAF and matrix metalloproteinase, as well aspro-coagulant tissue factors (for recent review see (28)). For thesereasons, the formation of platelet-leukocyte aggregates is targeted forthe therapeutic intervention (for reviews see (28,36)). The lipidomicsinvestigation indicated that 10,17-diHDHEA and 15-HEDPEA were two majorDHEA-derived products produced by isolated human PMN. Thus the actionsof these compounds were assessed in PAF stimulated platelet-leukocyteaggregate formation. Both 10,17-diHDHEA and 15-HEDPEA were potentsignals and, at concentrations as low as 10 pM, each decreased 100 nMPAF stimulated platelet-monocyte aggregate formation ˜30% in human wholeblood (FIG. 5a ). The 10,17-diHDHEA also decreased PAF stimulatedplatelet-PMN aggregates by 25%-35% (FIG. 5b ). For comparison, theprecursor DHEA did not significantly inhibit formation ofplatelet-leukocyte aggregates within this dose range (FIG. 5a and 5b ).

Formation of platelet-leukocyte aggregates depends mostly on the

activation of platelets (37). Along these lines, 10,17-diHDHEA (1.0 pMto 100 nM) blocked P-selectin surface expression of PAF stimulatedplatelets (FIG. 5c ), suggesting that 10,17-diHDHEA actions were atleast partially achieved via reductions in P-selectin mobilization andsurface appearance-related platelet activation. The present resultsdemonstrate that DHEA metabolic oxygenation generated potent moleculesthat reduce platelet activation and platelet-leukocyte aggregateformation in human whale blood.

Ischemia/reperfusion or reflow injury is the major cause of organ injurypost-myocardial infarction, stroke, surgery, and organ transplantationinjury and involves platelet and PMN activation (24). In this setting,neutrophils play critical roles in initiation of reperfusion or reflowinjury and in consequent tissue damage. Hence, prevention of PMNactivation or accumulation in ischemia organ reduces tissue injury afterreperfusion (24,38). The present results obtained from chemotaxisscreening might serve as useful benchmarks for searching/selectingpotential protective mediators for ischemia/reperfusion injury. In thisregard, 15-HEDPEA, which effectively stopped PMN chemotactic migration,was next evaluated in the mouse ischemia/reperfusion second organ injuryinitiated by hind limb occlusion. Indeed, 15-HEDPEA at 1 μg/mouse wasorgan protective, decreasing PMN infiltration in lung by ˜50%. It isnoteworthy that aberrant and excessive leukocytic infiltration is alsoassociated with other diseases, including arthritis and psoriasis(39,40). Of interest, Kim et al. recently reported that DHEA promotesdevelopment of hippocampal neurons (41).

In summation, lipidomic investigation of DHEA functional metabolomeuncovered a series of novel oxygenated products that 1) are potent CB2agonists, 2) regulate single-cell PMN chemotactic responses, 3) modulateplatelet-leukocyte interaction in whole blood, and 4) are organprotective. In view of the role of lipid mediators in inflammation andits resolution as well as hemostasis(7), the present new DHEA metabolomedocumented herein may serve as a counter-regulatory system in neuraltissues and those rich in DHEA as well as in administration of DHA (42)to regulate leukocyte-mediated tissue damage.

Materials and Methods

Material—LC grade solvents were purchased from Fisher Scientific(Pittsburgh, Pa.). Phenomenex Luna C18 (150 mm×2 mm×5 μm) column andStrata-X solid phase extraction columns were purchased from Phenomenex(Torrance, Calif.). Soybean lipoxygenase, human hemoglobin, human serumalbumin (HSA), d-4 MeOH, Hanks' balanced salt buffer (HBSS) werepurchased from Sigma-Aldrich (Milwaukee, Wis.).N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) was purchased fromPierce (Rockford, Ill.), P-selectin was purchased from R&D Systems(Minneapolis, Minn.). PE-conjugated mouse anti-human CD62P and FITC-CD41anti-human were purchased from BD Biosciences (Rockville, Md.).FITC-conjugated mouse anti-human CD14, mouse anti-human CD16,Cy5-conjugated mouse anti-human CD3, and mouse anti-human CD20 were allpurchased from Pharmingen (San Jose, Calif.). 17-HDHA standard wasprepared from DHA and soybean lipoxygenase (8,19). Doeosahexaenoylethanolamide (DHEA) was custom synthesized by Dr. Piomelli's group atUC-Irvine or purchased, as was 15(S)-HETE ethanolamide (15(S)-HAEA))from Cayman Chemical (Ann Arbor, Mich.).

Animals—All animals used in the present study were male FVB mice(Charles River Laboratories) that were 6-8 wk-old (weighing 20-25 g).They were maintained in a temperature and light-controlled environment,and had unlimited access to water and food (laboratory standard rodentdiet 5001 (Lab Diet)), containing 1.5% EPA, 1.9% DHA of total fattyacids. Experiments were performed in accordance with the Harvard MedicalSchool Standing Committee on Animals guidelines for animal care(Protocol 02570).

RP-HPLC—Liquid chromatographic analyses and separations were performedusing an Agilent 1100 series high performance liquid chromatography(HPLC) system (Agilent, Santa Clara, Calif.) equipped with G1379Adegasser, G1312A binpump and G13158 UV diode array detector. HPLCanalyses were carried out using a Phenomenex C18 column (150 mm×2 mm×5μm) with the mobile phase of 0.2 ml/min flow rate (methanol: water,70:30 v/v from 0 to 18 min, then ramped to 100% methanol from 18 to 35min). Compound isolations/purifications were carried out using a BeckmanODS column (10 mm×250 mm×5 μm) with the mobile, phase flow rate at 4ml/min (methanol: water, 70:30 v/v from 0 to 18 min, then ramped to 100%methanol from 18 to 35 min).

Lipidomics MS-MS Analyst—Sample analyses were carried out using a massspectrometer (Qstar XL quadrupole TOP hybrid mass spectrometer (AppliedBiosystems, Foster City, Calif.) equipped with two Shimazu LC20AD HPLCpumps (Shimazu, Columbia, Md.) and Agilent G1315B UV diode arraydetector (Agilent, Santa Clara, Calif.), For routine analyses, sampleswere extracted using C-18 cartridge as in (19) and injected to aPhenomenex C18 column (150 mm×2 mm×5 μm), and the mobile phase(methanol: water; 70:30 v/v from 0 to 18 min, then ramped to 100%methanol from 18 to 35 min) was eluted at a 0.2 ml/min flow rate and UVdetector scanned from 200 to 400 nm before samples entered the MS-MS.GC-MS analysis was carried out as in (9), Samples were injected in 2.5μL hexane.

Preparation of Oxygenated DHEA Products—DHEA (12.5 mg) was suspended in0.05 M borate buffer (250 ml, pH=9.3) at 4° C., and 160 kilounitssoybean LOX (type VI, 640 kilounits total, 701 kilounits/mg protein, 3.6mg of protein/ml) was added at 0, 2, 4, and 6 min. The incubation wasmonitored using UV spectrometer (Agilent, Santa Clara, Calif.).Incubations were treated with NaBH4 before extraction two times with 200ml ether. The organic layers were combined, washed twice with 100 mldd-H2O, taken to dryness under nitrogen flow, and subjected topreparative HPLC isolation monitoring online UV at 235 nm, 245 nm, and270 nm, for isolation of 4,17-diHDHEA, 7,17-diHDHEA, and 14,17-diHDHEArespectively. The corresponding fraction was collected, dried undernitrogen and resuspended in methanol. Preparation of each compound wasconfirmed using GC-MS or LC-MS-MS before further investigation.

Preparation of HEDPEA—Human hemoglobin (400 mg) was added to 17-HpDHEA(2.75 mg) suspended in 25 ml phosphate buffer (0.1 M, pH 7.3, 37° C.)and vortexed (5 min). The incubations were carried out at 37° C. for 6min, and then diluted with ddH₂O to 100 ml and extracted twice with 150ml ether. The organic layer was combined and washed twice with 100 mldd-H₂O. The crude product was taken to dryness under nitrogen flow andthen isolated by preparative HPLC isolation. The fractions were isolatedand collected monitoring UV absorbance at 215 nm. Each fraction wascollected, taken to dryness under nitrogen flow, subjected to LC-MS-MSand/or NMR analysis, or derivatized with BSTFA and then subjected toGC-MS analysis.

Receptor-Ligand Interactions—Receptor activation with the CB2 betaarrestin system was carried out essentially as in (20,21), HEK cellsstably overexpressing human CB2 receptor tagged with Pro-link and EAlabeled beta-arrestin (Discoverx, Fremont Calif.) were plated at 20,000cells/well of a 96 well plate. Forty-eight hours post plating, cellswere incubated with compounds at concentrations from 1 pM to 100 nM for1 h in serum free DMEM at 37° C. Ligand receptor interaction wasdetermined by measuring chemiluminescence using the Pathhunter EFCdetection kit (Discoverx, Fremont, Calif.), generated upon coupling ofthe EA labeled beta arrestin with the Pro-Link tagged receptor, with aplate reader (Envision, Perkin Elmer, Santa Clara, Calif.),

PMN Isolation and Incubations—Human whole blood was collected (Brighamand Women's Hospital protocol 88-02642) and PMNs were isolated as inrefs. (8,9,11). PMNs (2×10⁶) suspended in 1 ml DPBS+/+ with 0.2% bovineserum albumin (Sigma) were incubated with 5 μg of HPLC isolated17-HpDHEA or DHEA, alone or with Zymosan A (100 μg/ml) for 30 min at 37°C., and incubations were stopped with 2 volumes of ice-cold methanol.The mixture was kept in −20° C for at feast 2 h to precipitate proteins,and then taken for C18 solid phase extraction and analysis.

Leukocytic Chemotaxis Screening of DHEA Metabolites with MicrofluidicChamber—The fabrication and surface modification of the microfluidicdevices were prepared as in refs. (9.22). Whole blood (5-10 μl) dilutedin HBSS (1:10, v/v) was introduced into the chemotaxis chamber via acell inlet and neutrophils were captured along the chamber viaP-selectin tethering. Next, the transversal gradient of IL-8 (0-10 nM)was introduced to the chemotaxis chamber. After fifteen minutes, novelDHEA metabolites (at a uniform concentration) were introduced to thechemotaxis chamber from the second gradient generator network, and 10 nMIL-8 gradient was maintained. Single-cell neutrophil chemotaxis wasrecorded using microscopy (Nikon, Eclipse E600) equipped with a videocamera (Diagnostic, RT Slider) and subject to analysis using ImageJsoftware (9).

PAF-Stimulated Platelet-leukocyte Aggregate Formation—Whole blood wasincubated with either vehicle, HPLC isolated 10,17-diHDHEA or 15-HEDPEA(0.01-100 nM.) for 15 min at 37° C. with intermittent mixing. Vehicle orPAF (100 nM, PAF C-16, Cayman Chemical, Arm Arbor, Mich.) was added foranother 30 min at 37° C. with intermittent mixing. Incubation wasstopped by ice-cold red blood cell lysis buffer (10 min at 4° C.). Cellswere collected using centrifugation (210 g, 5 min, 4° C.) then fixedwith 3% formalin (15 min, 4° C.). Cells were stained withFITC-anti-human CD41 (1:100, v/v) and PE-anti-human-CD62P (1:100, v/v)for 20 min at 4° C., and were analyzed using a flow cytometry and CellQuest software as in (23). Cellular composition within whole blood wasdetermined by forward and side scattering as well as cell-specificmarkers, anti-human-CD41 for platelets, anti-human-CD14 for monocytesand anti-human-CD16 for neutrophils.

Second Organ Reperfusion Injury—Murine hind limb vascular occlusion

second organ lung reperfusion injury was performed using 6- to8-week-old FVB male mice and carried out as in (24).

Statistical Analysis—The significance of difference between groups wasevaluated using the two-tailed Student's t-test. P values of less than0.05 were considered to be statistically significant

RESULTS Functional Metabolomics

a) LC-UV-MS-MS Identification of 17-HDHEA From Brain.

To investigate the potential endogenous generation of DHEA-derivedbioactive products, mouse brain was harvested, subject to solid phaseextraction (19), and resulting methyl formate fractions were taken forLC-UV-MS-MS-based metabolomics. Tandem mass fragmentations and online UVspectrum with characteristic λ_(max) at 237 nm are consistent with theproposed structure as shown in the inset of FIG. 1. Because of the lackof suitable functional groups for direct efficient ionization andanalysis of 17-hydroxy-4Z, 7Z, 10Z, 13Z, 15,19Z-docosahexaenoylethanolamide (17-HDHEA), its acetate adduct m/z446=[M+CH₃COOH—H] was targeted for analysis. The major tandem mass ionswere assigned as following: m/z 386=[M-H], 368=[M-H-H₂O], 281=[299-H₂O].The m/z 288 is consistent with fragmentation at Position 17 (see Table 1for numbering) (FIG. 1b ). Because of the limited quantities ofendogenous 17-HDHEA produced in brain tissue, further analyses and invitro enzymatic preparation were carried out by incubating DHEA with15-LOX followed by reduction with NaBH₄(see Methods). Endogenous17-HDHEA and the enzymatically prepared compound in vitro gaveessentially the same LC retention times and tandem mass fragmentationsusing LC-MS-MS (see Supplemental FIG. 1). To assess their production byhuman and mouse tissues, DHEA was also incubated with isolated human PMNor whole mouse brain because DHEA is enriched in this tissue.LC-MS-MS-based targeted lipidomics indicated the production of a novelseries of oxygenated DHEA (Table 1).

b) Decoding Metabolomics Using Microfluidic Chambers.

In parallel to structure elucidation, chemotactic screening of HPLCisolated DHEA metabolites obtained from mouse brain was carried oututilizing microfluidic chamber (FIG. 1c ). After IL-8 (0-10 nM gradient)was introduced to the main channel of the microfluidic device,P-selectin tethered leukocytes rapidly migrated along the IL-8chemotactic gradient at an average rate of 2.3 μm/min. After 15 min, themixture of metabolites was infused into the microfluidic main channelwhile an IL-8 (0-10 nM) gradient was maintained (FIG. 1c , left panel).Human PMN chemotaxis was dramatically reduced (p<0.01) upon the additionof the brain metabolite mixture, whereby average human PMN chemotaxisvelocity dropped from 2.3 μm/min to ˜0.7 μm/min (FIG. 1C , middlepanel). This decrease in chemotaxic velocity was maintained even afterthe gradient was switched back to IL-8. These results indicated that thebrain metabolites contained bioactive components that stopped PMNchemotaxis.

c) LC-UV-MS-MS and GC-MS-based metabolomics of DHEA.

Results from this screening uncovered that at least one bioactiveproduct was present among the mixture of DHEA metabolites; thus, wepursued the metabolic fates of DHEA and 17-HpDHEA/ 17-HDHEA identifiedin mouse brain (FIG. 1b ) using LC/UV/MS/MS-based lipidomics. As with17-HDHEA, acetate adducts of potential DHEA derived metabolites[M+CH₃COOH—H] were targeted for tandem mass analysis (Table 1). Theseresults demonstrated the presence and production of novel products inthe DHEA metabolome.

Incubations of isolated human PMNs with DHEA or 17-HpDHEA led to thegeneration of 17-HDHEA, 4,17-diHDHEA, 10,17-diHDHEA and 15-HEDPEA. Humanhemoglobin, which can be liberated upon tissue damage (25), wasincubated with 17-hpDHEA, gave 13-HEDPEA and 15-HEDPEA as prominentproducts, as well as 17-HDHEA (Table 1). Mouse brain homogenates withDHEA also produced 17-HDHEA and 4,17-diHDHEA as major products withsmaller amounts of 7,17-diHDHEA, 10,17-diHDHEA and 15-HEDPEA. The onlineUV and tandem mass spectra for 4,17-diHDHEA are shown in FIG. 2a and 2b. The adduct parent ion, analyte parent ion and the ions resulted fromneutral loss are m/z 462=[M+CH₃COOH—H], 402=[M-H], 384=[M-H—H₂O],366=[M-H-2H₂ 0], which are common signature ions for alldihydroxy-containing DHEA products. The ions m/z 333, 315=[333-H₂O],304, 286=[304-H₂O] were assigned as diagnostic sons for fragmentationsat Position 17. Fragmentations at Position 4 can lead to m/z 144, 2,57and 239=[257-H₂O]. Its UV spectrum displayed characteristic maximumabsorbance at 238 nm, which was consistent with the presence of twoseparated conjugated diene structures in this compound. As shown in FIG.2c and 2d , diagnostic ions m/z 304, 286=[304-H2O]; 184, 156,corresponded to the fragmentations at Positions 7 and 17 of 7,17-diHDHEArespectively (see Table 1 for numbering). The LTV spectrum of thecompound displayed maximum absorbance, λ_(max), at 246 nm (26),consistent with the presence of two diene structures separated by amethylene group. For 10,17-diHDHEA, m/z 333, 315=[333-H2O], 304,285-[304-H₂O], 196 came from fragmentations at Positions 10 and 17 asshown in FIG. 2e and 2f . The presence of a conjugated triene structurein 10,17-diHDHEA was confirmed by the characteristic UV spectrum with at270 nm. Tandem mass spectrum, of 13-HEDPEA is shown in Supplemental FIG.2a with signature fragmentation ions m/z 320,304,286=[304-H2O], 236.GC/MS was also utilized for additional structural analysis with13-HEDPEA and 15-HFDPFA that confirmed the original tandem MSassignments shown in Supplemental FIG. 2c and 2d . The C-value for13-HEDPEA was determined as 32.1±0.2 (Supplemental. FIG. 2e ) and for15-HEDPEA was 33.7±0.2 (Supplemental FIG. 2f ).

In order to determine concentrations, as well as to further confirmstructures, HPLC isolated 13-HEDPEA and 15-HEDPEA were characterizedusing proton NMR (¹H NMR). The chemical shift assignments are shown inSupplemental Tables 2a and 2b, respectively. For 15-HEDPEA, the protonat Position 15 (H-15) displayed two distinct chemical shifts, which willbe discussed later. Because of limited amounts of materials and the lackof informative UV chromophores present in these compounds, NMRspectroscopy was also used for quantitation using 17-HDHA as an internalstandard with known concentrations. The NMR quantitated compounds werethen used as standards for HPLC quantitation monitoring UV chromatogramat 210 nm or LC-tandem mass profiling (see Methods for further details).

Human PMN single cell chemotactic functional screening.

HPLC isolated dioxygenated DHEA products were individually screened fordirect PMN actions using microfluidic chambers. Infusion of isolated15-HEDPEA at 10 nM to the main channel stimulated changes in morphologyand chemotaxis of PMN in the IL-8 gradient and stopped further PMNmigration after ˜4 min (FIG. 3a ). For direct comparison, PMN chemotaxisvelocity did not change with time with the IL-8 gradient (SupplementalFIG. 3a ). At 10 nM, 4,17-diHDHEA (FIG. 3b ), 7,17-diHDHEA or10,17-diHDHEA did not significantly regulate chemotaxis (SupplementalFIG. 3b,c ), while at higher concentrations, e.g. 10 μM, 10,17-diHDHBArapidly stopped PMN chemotaxis (FIG. 3c ). These results indicate that15-HEDPEA is the most potent of this series in regulating human PMNshape change and motility.

Cannabinoid (CB) receptor activation.

Since AEA exerts a wide range of bioactions via activating cannabinoidreceptors) (14,27), It was next tested whether DHEA, 10,17-diHDHEA or15-HEDPEA also activated CB receptors. To this end, recombinant human CBreceptors were overexpressed in a beta-arrestin .system as described inExperimental Procedures. AEA was used for direct comparison as a knownagonist. FIG. 4 shows the dose response of CB 1 and 2 with eachcompound. Activation of CB2 by AEA gave EC₅₀˜1.1×10⁻¹⁰ M and DHEA9.8×10⁻⁹ M. For comparison, EC₅₀ for metaboiically oxygenated products,10,17-diHDHEA and 15-HEDPEA, were 3.9×10⁻¹⁰ M and 1.0×10⁻¹⁰ Mrespectively. These results demonstrate enzymatic oxidation productsfrom DHEA are activators of CB2 receptors, and that 10,17-diHDHEA and15-HEDPEA also activated CB1 receptors but required much higherconcentrations (FIG. 4a ). By comparison, 15(S)-HAEA, the oxygenatedproduct of AEA, did not stimulate CB2 receptors in this dose range, CB2receptor-ligand interactions were confirmed with dose response of CB2specific antagonist AM630. When incubated with GPCR CB2 over-expressedcells, AM63G inhibited activation stimulated with 15-HEDPEA (10 nM) andAEA (10 nM), used here for a known positive and direct comparison (FIG.4e and 4f ). AM630 also inhibited GPCR CB2 interaction with10,17-diHDHEA at higher concentration (n=3, data not shown).

DHEA products reduced platelet-leukocyte aggregate formation in humanwhole blood.

Platelet-leukocyte interactions play important roles in hemostasia,thrombosis, and inflammation (for recent review see (28) and referenceswithin). At concentrations as low as 10 pM, 10, 17S-diHDHEAor 15-HEDPEAdecreased PAF-(100 nM) stimulated platelet-monocyte aggregate formationin human whole blood by ˜30% (FIG. 5a,b ). The inhibitory action of10,17-diHDHEA displayed a bell-shaped dose response and reached maximumreduction at ˜40% with 100 pM. Formation of PMN-platelet aggregates withPAF (100 nM) was also inhibited by 10,17-diHDEA at concentrations as lowas 10 pM, as was the surface expression of P-selectin on platelets inwhole blood (FIG. 5d ). By comparison, the precursor DHEA (unoxidized)was not active in this dose range (FIG. 5a,b ).

Organ protection in ischemia/reperfusion injury.

Since 15-HEDPEA displayed potent bioactions with human PMN at singlecell level (FIG. 3) and in human whole blood (FIG. 5), it was nextquestioned whether it had protective actions in vivo in murine hind limbischemia (1 h) and second organ reperfusion (2 h) injury (24). Indeed,following reperfusion, 15-HEDPEA significantly reduced lung PMNaccumulation in mice and associated lung injury at 1 μg/mouse,(Supplemental FIG. 4) (˜50% reduction compared to vehicle; P<0.05).

SUPPLEMENTAL INFORMATION Supplemental Table 1, Structures, LC-MS andGC-MS Fragmentations, and UV Xmax for Novel DHEA Metabolites IdentifiedUsing Mediator-based Lipidomics

SUPPLEMENTAL TABLE 1 Structures, LC-MS and GC-MS fragmentations, and UVλmax for novel DHEA metabolites identified using mediator-basedlipidomics. LC LC-MS reten- major/ PMN + tion diagnostic UV DHEA MouseTrivial time fragmentions λ max or Hgb + brain + Structure† Name (min)(m/z) (nm) HpDHEA HpDHEA DHEA

13- HEDPEA 20.0 462(M + AcOH—H), 402(M—H), 384, 366, 320, 304, 286, 236.no conju- gated sys- tem iden- tified trace yes not de- tected

16-GS- 17- HDHEA 691(M—H), 673, 359, 562, 306, 272, 254. 278 PMN withHpDHEA and GSH

4, 20- diHDHEA 462(M + AcOH—H), 402(M—H), 384, 366, 257, 239, 144. 236 Amajor product of incubation of mouse brain and DHEA †Stereochemistriesare tentatively assigned and the action of these products will bereported separately.

SUPPLEMENTAL TABLE 2a NMR assignments of 15-hydroxy-16(17)-epoxy- DHEA.Proton position δ (mult, J/Hz) H-1 3.59 (t, J = 5.8) H-2 3.28 (t, J =5.8) H-5 2.25 (t, J = 7.50 H-6 2.38 (m) H-7, H-8, H-10, H-11, H-13,H-14, H-22, 5.3-5.5 (m) H-23 H-9, H-12, H-15, H-19, H-20, H-21,2.80-2.95 (m) H-16, H-17 5.50-5.60 (m) H-18 4.23 (m) and 4.45 (m) H-244.45 (m) H-25 0.96 (td, J = 7.4, 1.4)

SUPPLEMENTAL TABLE 2b NMR assignments of 13-hydroxy-16(17)-epoxy-docosapentaenyolethanolamide.

Proton position δ(mult, J/Hz) H-1 3.58 (t, 5.9) H-2 3.28 (t, 5.8) H-52.25 (t, 7.2) H-6, H-9, H-12 2.35 (m) H-7, H-8, H-10, H-11, H-13, H-14,H-22, 5.35-5.50 (m) H-23 H-16 3.9 (m) H-17 5.58 (m) H-18 5.95 (dd, 15.5,5.7) H-19 3.19 H-20, H-21 2.08 (m) H-24 2.08 (m) H-25 0.97 (t, 7.5)

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The inventions illustratively disclosed herein suitably may be practicedin the absence of any element which is not specifically disclosedherein. Additionally, the inventions illustratively disclosed herein maybe practiced in the absence of any element disclosed herein.

Although the present invention has been described with reference topreferred embodiments, persons skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention. All references cited throughout thespecification, including those in the background, are incorporatedherein in their entirety. Those skilled in the art will recognize, or beable to ascertain, using no more than routine experimentation, manyequivalents to specific embodiments of the invention describedspecifically herein. Such equivalents are intended to be encompassed inthe scope of the following claims.

1. A compound comprising one of the formulae selected from (I) through(VIIIa):

wherein each of P₁ and P₂ individually, if present, is a protectinggroup or a hydrogen atom; wherein

is a double bond if present; wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(O)NR^(c)R^(c)—OH, —C(O)H, —C(NH)NR^(c)R^(c), —C(S)H, —C(S)OR^(d) or—C(S)NR^(c)R^(c); OR —CN; each R^(a), is independently hydrogen, (C1-C6)alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylalkyl,(C5-C10) aryl, phenyl, (C6-C16) arylalkyl, benzyl, 2-6 memberedheteroalkyl, 3-8 membered cycloheteroalkyl, morpholinyl, piperazinyl,homopiperazinyl, piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10membered heteroaryl or 6-16 membered heteroarylalkyl; each R^(c), isindependently a protecting group or R^(a), or, alternatively, each R^(c)is taken together with the nitrogen atom to which it is bonded to form a5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally haveone or more of the same or different additional heteroatoms and whichmay optionally be substituted with one or more of the same or differentR^(a) or suitable R^(b) groups; each R^(b) is independently ═O, —OR^(d),(C1-C3) haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d),—NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂—NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —OC(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a),—C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c),—OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(a), —[NHC(O)]_(n)R^(d),—[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d),—[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) or —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c); each n,independently is an integer from 0 to 3; and each R^(d), independentlyis a protecting group or R^(a), or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1, wherein P₁ and P₂ are both hydrogenatoms.
 3. The compound of claim 1, wherein Z is —C(O)NR^(c)R^(c)—OH. 4.The compound of claim 3, wherein one R^(c) is H and the second R^(c) isethyl,
 5. The compound of claim 1, wherein the hydrogen atom on one ormore hydroxyl containing carbon atoms is substituted with an alkylgroup.
 6. The compound of claim 5, wherein the alkyl group is a methylgroup.
 7. The compound of claim 10, wherein Z is —C(O)OR^(d) and R^(d)of Z is a hydrogen atom.
 8. A method to treat inflammation,neurodegeneration, memory loss, neuroinflammation, reperfusion injury ortraumatic brain injury comprising the step of administering to anindividual in need thereof, an effective amount of the compound ofclaim
 1. 9. A method to treat neural development, fetal development,homeostasis, tissue remodeling, or wound repair comprising the step ofadministering to an individual in need thereof, an effective amount ofthe compound of claim
 1. 10. The compound of claim 1, further comprisinga pharmaceutically acceptable carrier.
 11. A purified compoundcomprising one of the formulae (I) through (VIIIa):

wherein each of P₁ and P₂ individually, if present, is a protectinggroup or a hydrogen atom; wherein

wherein Z is —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(O)NR^(c)R^(c)—OH, —C(O)H,—C(NH)NR^(c)R^(c), —C(S)H, —C(S)OR^(d) or —C(S)NR^(c)R^(c); OR —CN; eachR^(a), is independently hydrogen, (C1—C6) alkyl, (C3—C8) cycloalkyl,cyclohexyl, (C4-C11) cycloalkylalkyl, (C5-C10) aryl, phenyl, (C6-C16)arylalkyl, benzyl, 2-6 membered heteroalkyl, 3-8 memberedcycloheteroalkyl, morpholinyl, piperazinyl, homopiperazinyl,piperidinyl, 4-11 membered cycloheteroalkylalkyl, 5-10 memberedheteroaryl or 6-16 membered heteroarylalkyl; each R^(c), isindependently a protecting group or R^(a), or, alternatively, each R^(c)is taken together with the nitrogen atom to which it is bonded to form a5 to 8-membered cycloheteroalkyl or heteroaryl which may optionally haveone or more of the same or different additional heteroatoms and whichmay optionally be substituted with one or more of the same or differentR^(a) or suitable R^(b) groups; each R^(b) is independently ═O, —OR^(d),(C1-C3) haloalkyloxy, —OCF₃, ═S, —SR^(d), ═NR^(d), ═NOR^(d),—NR^(c)R^(c), halogen, —CF₃, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —OC(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —C(NR^(a))NR^(c)R^(c), —C(NOH)R^(a),—C(NOH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c),—OC(NH)NR^(c)R^(c), —OC(NR^(a))NR^(c)R^(c), —[NHC(O)]_(n)R^(d),—[NR^(a)C(O)]_(n)R^(d), —[NHC(O)]_(n)OR^(d), —[NR^(a)C(O)]_(n)OR^(d),—[NHC(O)]_(n)NR^(c)R^(c), —[NR^(a)C(O)]_(n)NR^(c)R^(c), —[NHC(NH)]_(n)NR^(c)R^(c) or —[NR^(a)C(NR^(a))]_(n)NR^(c)R^(c); each n,independently is an integer from 0 to 3; and each R^(d), independentlyis a protecting group or R^(a), or a pharmaceutically acceptable saltthereof.
 12. The purified compound of claim 11, wherein P₁ and P₂ areboth hydrogen atoms.
 13. The purified compound of claim 11, wherein Z is—C(O)OR^(d) and R^(d) of Z is a hydrogen atom.
 14. The purified compoundof claim 11, wherein one R^(c) is H and the second R^(c) is ethyl. 15.The purified compound of claim 11, wherein Z is —C(O)NR^(c)R^(c)—OH. 16.The purified compound of claim 11, wherein the hydrogen atom on one ormore hydroxyl containing carbon atoms is substituted with an alkylgroup.
 17. The purified compound of claim 16, wherein the alkyl group isa methyl group.
 18. The compound of claim 11, further comprising apharmaceutically acceptable carrier.
 19. A method to treat inflammation,neurodegeneration, memory loss, neuroinflammation, reperfusion injury ortraumatic brain injury comprising the step of administering to anindividual in need thereof, an effective amount of the compound of claim11.
 20. A method to treat neural development, fetal development,homeostasis, tissue remodeling, or wound repair comprising the step ofadministering to an individual in need thereof, an effective amount ofthe compound of claim 11.